Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum, and are among the most potent toxins known. These toxins are a well-recognized source of food poisoning, often resulting in serious harm or even death of the victims. There are seven structurally related botulinum neurotoxins or serotypes (BoNT/A-G), each of which is composed of a heavy chain (˜100 KD) and a light chain (˜50 KD). The heavy chain mediates toxin entry into a target cell through receptor-mediated endocytosis. Once internalized, the light chain is translocated from the endosomal vesicle lumen into the cytosol, and acts as a zinc-dependent protease to cleave proteins that mediate vesicle-target membrane fusion (“substrate proteins”).
These BoNT substrate proteins include plasma membrane protein syntaxin, peripheral membrane protein SNAP-25, and a vesicle membrane protein synaptobrevin (Syb). These proteins are collectively referred to as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Cleavage of SNARE proteins blocks vesicle fusion with plasma membrane and abolishes neurotransmitter release at neuromuscular junction. Among the SNARE proteins, syntaxin and SNAP-25 usually reside on the target membrane and are thus referred to as t-SNAREs, while synaptobrevin is found exclusively with synaptic vesicles within the synapse and is called v-SNARE. Together, these three proteins form a complex that is thought to be the minimal machinery to mediate the fusion between vesicle membrane and plasma membrane. BoNT/A, E, and C cleave SNAP-25, BoNT/B, D, F, G cleave synaptobrevin (Syb), at single but different sites. BoNT/C also cleaves syntaxin in addition to SNAP-25.
Due to their threat as a source of food poisoning, and as bioterrorism weapons, there is a need to sensitively and speedily detect BoNTs. Currently, the most sensitive method to detect toxins is to perform toxicity assay in mice. Such methods, however, entail considerable expense and are subject to regulations related to animal testing.
As a result, there is a growing interest in developing alternatives to animal-based methods for BoNT characterization. An attractive alternative is the use of cell-based assays, which maintain the receptor-based internalization and subsequent cleavage of the BoNT molecule that is generally absent from conventional in vitro assays. Such cell-based assays utilize cells that express constructs that are responsive to the BoNT, in some instances utilizing Förster resonance energy transfer (FRET) and in other instances utilizing non-FRET methods to provide fluorescence useful for the detection and characterization of BoNTs. Examples can be found in United States Patent Application No. 2004/0,191,887 (to Chapman), United States Patent Application No. 2006/0,134,722 (to Chapman), U.S. Pat. No. 7,208,285 (to Steward), U.S. Pat. No. 7,183,066 (to Fernandez-Salas), and United States Patent Application No. 2011/0,033,866 (to Atapattu), each of which is incorporated herein by reference in their entirety. For some applications, however, the sensitivity of such cell-based methods can be lacking. For example, United States Patent Application No. 2006/0,134,722 (to Chapman) discloses that EC50 value of cell based FRET assay to detect BoNTs is in the ≥10 pM range.
International Patent Application No. WO 2014/060373 (to Eisele) reported enhancement of the sensitivity of cells to intoxication with botulinum toxin by allowing certain tumor cells that had been primed for differentiation into neuronal cells to differentiate in a low osmolarity differentiation media for several days to several weeks prior to exposure to the toxin. Sensitivity was determined by lysis of the treated cells followed by a Western blot method directed towards SNAP-25. The utility of Western blotting as a quantitative method is considered debatable, however, and no data demonstrating the statistical significance of the reported differences was provided.
Although some success has been demonstrated in applying FRET assays to detection of BoNTs, the sensitivity of FRET assay to BoNTs has been still undesirable for many purposes. As few as 40 nanograms of BoNT is a lethal dose for most people, and samples suspected to contain BoNTs are often are prior to application to the test process. It is, therefore, strongly desirable to have methods that detect low concentrations of BoNT.